I. Field of the Invention
This invention is directed to particular tissue plasminogen activator (t-PA) variants, to methods for preparing these variants, and to methods and compositions utilizing the variants in pharmaceutical compositions. Specifically, this invention relates to t-PA variants with modified amino acid sequences, including substitutions, within at least the kringle-1 or kringle-2 domains of t-PA that result in the variants having a decreased rate of clearance as compared to wild-type t-PA.
II. Description of Background and Related Art
Plasminogen activators are enzymes that cleave the peptide bond of plasminogen between amino acid residues 561 and 562, converting it to plasmin. Plasmin is an active serine proteinase that degrades various proteins including fibrin. Several plasminogen activators have been identified including streptokinase (a bacterial protein), urokinase (synthesized in the kidney and elsewhere and originally extracted from urine), and human tissue plasminogen activator, termed t-PA (produced by the cells lining blood vessel walls).
The mode of action of each of these plasminogen activators is somewhat different. Streptokinase forms a complex with plasminogen or plasmin generating plasminogen-activating activity, urokinase cleaves plasminogen directly, and t-PA interacts with both plasminogen and fibrin for optimal activity.
Due in part to its high fibrin specificity and potent ability to dissolve blood clots in vivo, t-PA has been identified as an important new biological pharmaceutical for treating vascular diseases such as myocardial infarction.
A substantially pure form of t-PA was first produced from a natural source and tested for in vivo activity by Collen et al., U.S. Pat. No. 4,752,603 issued Jun. 21, 1988 (see also Rijken et al., J. Biol. Chem., 256:7035 [1981]). Pennica et al. (Nature, 301:214 [1983]) determined the DNA sequence of t-PA and deduced the amino acid sequence from this DNA sequence (see U.S. Pat. No. 4,766,075 issued Aug. 23, 1988).
Human native t-PA has potential N-linked glycosylation sites at amino acid positions 117, 184, 218, and 448. A high mannose oligosaccharide is present at position 117 and a complex oligosaccharide is present at positions 184 and 448. Sites 117 and 448 appear to always be glycosylated, while site 184 is thought to be glycosylated in about fifty percent of the molecules. The partial glycosylation pattern at position 184 may be due to site 184 being situated in an unexposed region of the molecule. The t-PA molecules that are glycosylated at position 184 are termed Type I t-PA, and the molecules that are not glycosylated at position 184 are termed Type II t-PA. Position 218 has not been found to be glycosylated in native t-PA.
Research on the structure of t-PA has identified the molecule as having five domains. Each domain has been defined with reference to homologous structural or functional regions in other proteins such as trypsin, chymotrypsin, plasminogen, prothrombin, fibronectin, and epidermal growth factor (EGF). These domains have been designated, starting at the N-terminus of the amino acid sequence of t-PA, as the finger (F) domain from amino acids 1 to about 44, the growth factor (G) domain from about amino acids 45 to 91 (based on homology with EGF), the kringle-1 (Kl) domain from about amino acids 92-173, the kringle-2 (K2) domain from about amino acids 180 to 261, and the serine protease (P) domain from about amino acid 264 to the carboxyl terminus at amino acid 527. These domains are situated essentially adjacent to each other, and some are connected by short "linker" regions. These linker regions bring the total number of amino acids of the mature polypeptide to 527, although three additional residues (Gly-Ala-Arg) may be found at the amino terminus and are probably the result of incomplete precursor processing of the molecule.
Each domain is believed to confer certain biologically significant properties on the t-PA molecule. The finger domain is thought to be important in the high binding affinity of t-PA to fibrin. This activity appears to be important for the high specificity that t-PA displays with respect to clot lysis at the locus of a fibrin-rich thrombus. The kringle-1 and kringle-2 domains also appear to be associated with fibrin binding and with the ability of fibrin to stimulate the activity of t-PA. The serine protease domain is responsible for the enzymatic activity of t-PA which results in the conversion of plasminogen to plasmin. The t-PA molecule is often cleaved between position 275 and position 276 (located in this serine protease domain) to generate the 2-chain form of the molecule.
Natural t-PA has a plasma half-life of about six minutes or less when administered to patients in therapeutically effective amounts. In certain situations, a six-minute half-life is desirable, as for example, in aggressive therapy of a life-threatening disease such as myocardial infarction or pulmonary embolism. In these high-risk situations, patients who have significant or unrecognized potential for uncontrolled bleeding may be treated with t-PA. If such bleeding occurs, drug administration can be stopped and the causative t-PA levels will rapidly drop. Thus, treatment of these patients with a relatively short-lived form of t-PA is preferred.
Despite the profound advantages associated with natural t-PA as a clot-dissolving agent, it is not believed that the naturally occurring form of the protein necessarily represents the optimal t-PA agent under all circumstances. In some instances, such as treatment of deep vein thrombosis, treatment following reperfusion of an infarct victim, treatment of pulmonary embolism, or treatment using bolus injection, a t-PA molecule with a longer half-life and/or decreased clearance is desirable. Several variants of the wild-type t-PA molecule have been generated in attempts to increase half-life or decrease the clearance rate.
One method used to generate such t-PA variants has been to delete individual amino acids, partial domains, or complete domains from the molecule. For example, removal of part or all of the finger domain of t-PA as described in U.S. Patent Number 4,935,237 (issued Jun. 19, 1990) results in a molecule with decreased clearance, although it has substantially diminished fibrin-binding characteristics. Browne et al. (J. Biol. Chem., 263:1599 [1988]) deleted the region between amino acids 57 and 81 and found the resulting variant to have a slower clearance from plasma. Collen et al. (Blood, 71:216 [1988]) deleted amino acids 6-86 (part of the finger and growth domains) and found that this mutant had a half-life in rabbits of 15 minutes as compared with 5 minutes for wild-type t-PA. Similarly, Kaylan et al. (J. Biol. Chem., 263:3971 [1988]) deleted amino acids 1-89 and found that the half-life of this mutant in mice was about fifteen minutes as compared to about two minutes for wild-type t-PA. Cambier et al. (J. Cardiovasc. Pharmacol., 11:468 [1988]) constructed a variant with the finger and growth factor domains deleted and the three asparagine glycosylation sites abolished. This variant was shown to have a longer half-life than wild-type t-PA when tested in dogs. Variants with only the growth factor domain or the finger domain deleted have also been demonstrated to have decreased clearance rates in rabbits, guinea pigs and rats (Higgins and Bennett, Ann. Rev. Pharmacol. Toxicol., 30:91 [1990] and references therein).
Various deletions in the growth factor region have also been reported in the patent literature. See EPO Publication Number 241,208 (deletion of amino acids 51-87, and deletion of amino acids 51-173). See also EPO Publication Number 240,334 which describes the modification of mature, native t-PA in the region of amino acids 67-69 by deletion or substitution of one or more amino acids.
Another means to improve the clearance rate and/or half-life of t-PA has been to complex the t-PA molecule with a second molecule. For example, a t-PA-polyethylene-glycol conjugate has been reported to enhance the rate of clearance of t-PA, as reported in EPO 304,311 (published Feb. 22, 1989). A monoclonal antibody to t-PA has been reported to increase the functional half-life of t-PA in vivo without decreasing its activity (see EPO 339,505 published Nov. 2, 1989).
A variety of amino acid substitution t-PA variants have been evaluated for their ability to decrease the clearance rate or increase the half-life of t-PA. The variant R275E (where arginine at position 275 of native, mature t-PA was substituted with glutamic acid) has been shown to have a clearance rate of about two times slower than that of wild-type t-PA when tested in primates and rabbits (Hotchkiss et al., Thromb. Haemost., 58:491 [1987]). Substitutions in the region of amino acids 63-72 of mature, native t-PA, and especially at positions 67 and 68, have been reported to increase the plasma half-life of t-PA (see WO 89/12681, published Dec. 28, 1989).
Production of other substitution variants has focused on converting the glycosylation sites of t-PA to non-glycosylated sites. Hotchkiss et al. (Thromb. Haemost., 60:255 [1988]) selectively removed oligosaccharide residues from the t-PA molecule, and demonstrated that the removal of these residues decreased the rate of clearance of t-PA when tested in rabbits. Removal of the high mannose oligosaccharide at position 117 using the enzyme endo-.beta.-N-acetylglucosaminidase H (Endo-H) resulted in a rate of clearance that was decreased about two fold. Oxidation of nearly all oligosaccharide residues using sodium periodate resulted in a rate of clearance nearly three fold lower than wild-type t-PA. These researchers also generated the t-PA variant N117Q (wherein asparagine at position 117 of native, mature t-PA was substituted with glutamine) to prevent glycosylation at position 117. The clearance rate of this variant was lower than wild-type t-PA. See also EP 238,304 published Sep. 23, 1987 and EP 227,462 published Jul. 1, 1987.
An additional approach to produce t-PA variants with extended circulatory half-life and slower clearance has been to add glycosylation sites to the molecule. As examples of this approach, positions 60, 64, 65, 66, 67, 78, 79, 80, 81, 82, and 103 have been substituted with appropriate amino acids to create molecules with glycosylation sites at or near some of these residues (see WO 89/11531, published Nov. 30, 1989 and U.S. Ser. No. 07/480691, filed Feb. 15, 1990 now abandoned).
While some of the above cited work has resulted in generation of t-PA variants with increased half-life or decreased clearance rates, in many instances the activity, solubility, and/or fibrin-binding specificity of the molecule has been compromised. Thus, the known t-PA variants have not possessed optimal characteristics.
Accordingly, it is an object of this invention to prepare t-PA variants with decreased clearance rates that substantially retain biological activity, solubility and/or fibrin specificity. Production of t-PA variants with decreased clearance that possess any one or a combination of these characteristics will improve the therapeutic value and efficacy of t-PA. A further object of this invention is to produce t-PA variants with improved efficacy or pharmaceutical utility.